INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

Combined Mcl-1 and YAP1/TAZ inhibition for treatment of metastatic uveal melanoma.

Uveal melanoma is the most common intraocular tumor in adults, representing approximately 5% of all melanoma cases. Up to 50% of uveal melanoma patients develop metastases that are resistant to most of the commonly used antineoplastic treatments. Virtually all uveal melanoma tumors harbor activating mutations in GNAQ or GNA11 , encoding Gαq and Gα11, respectively. Constant activity of these proteins causes deregulation of multiple downstream signaling pathways including PKC, MAPK and YAP1/TAZ. While the importance of YAP1 signaling for the proliferation of uveal melanoma has recently been demonstrated, much less is known about the paralog of YAP1 transcriptional coactivator, named TAZ; however, similar to YAP1, TAZ is expected to be a therapeutic target in uveal melanoma. We performed a small-scale drug screen to discover a compound synergistically inhibiting uveal melanoma proliferation/survival in combination with YAP1/TAZ inhibition. We found that the combination of genetic depletion of YAP1/TAZ together with Mcl-1 inhibition demonstrates a synergistic inhibitory effect on the viability of uveal melanoma cell lines. Similarly, indirect attenuation of the YAP1/TAZ signaling pathway with an inhibitor of the mevalonate pathway, that is, the geranyl-geranyl transferase inhibitor GGTI-298, synergizes with Mcl-1 inhibition. This combination could be potentially used as a treatment for metastatic uveal melanoma.

Protein kinase inhibitor responses in uveal melanoma reflects a diminished dependency on PKC-MAPK signaling.

Uveal melanoma (UM) is a rare cancer arising from melanocytes in the uveal tract of the eye. Despite effective primary treatment, there is no approved therapy for metastatic UM and prognosis and survival remain poor. Over 90% of UM are driven by mutations affecting the Gα subunits encoded by the GNAQ and GNA11 genes. These mutations activate downstream and targetable signaling pathways, including the protein kinase C (PKC) cascade. PKC inhibitors have been used in clinical trials for metastatic UM but have shown limited efficacy. In this study, we examined the signaling and functional effects of two PKC inhibitors (AEB071 and IDE196) in a panel of UM cell models. In response to PKC inhibition, all UM cell lines showed potent suppression of PKC activity, but this was not sufficient to predict PKC inhibitor sensitivity and only two UM cell lines showed substantial PKC inhibitor-induced cell death. The differences in UM cell responses to PKC inhibition were not attributable to the degree or timing of PKC suppression or inhibition of the downstream mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) pathways. Instead, UM cell show complex, PKC-independent signaling pathways that contribute to their survival and resistance to targeted therapies.

Targeting GNAQ/11 through PKC inhibition in uveal melanoma.

Uveal melanoma is a rare malignancy affecting 5.1 patients/million per year with definitive treatment options of enucleation or radiation therapy to the primary tumor. Unfortunately, no FDA-approved systemic therapies exist for patients in the adjuvant or metastatic setting. Molecular profiling over the past decade has helped define uveal melanomas by characteristic mutations: GNAQ, GNA11, BAP1, SF3B1, and EIF1AX mutations. GNAQ/11 mutations are present in over 90% of patients with uveal melanoma and lead to signal transduction through G-protein coupled receptors to downstream growth factors. PKC inhibition has been an active area of investigation targeting this pathway specific to uveal melanoma. Several molecules have been developed and evaluated in clinical trials. Responses have been noted but clinical development has also yielded multiple toxicities and pathways of resistance limiting both breadth and durability of responses leading to combination therapy approaches. PKC inhibition remains an active and encouraging area of research to determine effective therapies for patients with uveal melanoma.

Gαq-protein carboxyl terminus imitation polypeptide GCIP-27 attenuates proliferation of vascular smooth muscle cells and vascular remodeling in spontaneously hypertensive rats.

Gq-protein is located at the convergent point in signal transduction pathways leading to vascular remodeling. The carboxyl terminus of Gα-subunit plays a vital role in G-protein-receptor interaction. The present study was designed to explore the effects of a synthetic Gαq carboxyl terminus imitation peptide, namely GCIP-27, on vascular smooth muscle cells (VSMC) in vitro and vascular remodeling in spontaneous hypertensive rats (SHR). Hyperplasia and hypertrophy of VSMC wre determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, [(3)H]-thymidine and [(3)H]-leucine incorporation, and [Ca(2+)](i) was measured with Fluo-3/AM staining. Systolic blood pressure (SBP), the ratio of media thickness to lumen diameter (MT/LD) of aorta, collagen content, and phospholipase C activity in aorta were measured in SHR. GCIP-27 (3-100 µg/l) significantly decreased proliferation activity, protein content, incorporation of [(3)H]-thymidine and [(3)H]-leucine, and [Ca(2+)](i) level in VSMC. SBP, MT/LD, collagen content, and phospholipase C activity in aorta of SHR were decreased significantly in GCIP-27 (7, 20, 60 µg/kg)-treated groups and losartan (6 mg/kg) group compared with vehicle group. In conclusion, GCIP-27 could inhibit vascular remodeling effectively in vitro and in vivo.

Inhibition of left ventricular remodelling in spontaneously hypertensive rats by G alpha q-protein carboxyl terminus imitation polypeptide GCIP-27 is not entirely dependent on blood pressure.

The G(q)-protein is located at the convergent point in the signal transduction pathway that leads to ventricular remodelling. In G-protein signalling pathways, the carboxyl terminus of the G(alpha)-subunit plays a vital role in G-protein-receptor interaction. The aim of the present study was to explore the effects of a synthetic G(alphaq) carboxyl terminus imitation peptide, namely GCIP-27, on left ventricular (LV) remodelling and blood pressure in spontaneous hypertensive rats (SHR). In the present study, 10, 30 or 90 microg/kg, i.p., GCIP-27 was administered for 8 weeks to SHR. In addition, another two groups of SHR were treated with either 6 mg/kg losartan or vehicle (saline). Wistar-Kyoto rats were used as controls. Systolic blood pressure (SBP) was measured using the standard tail-cuff method once every 2 weeks. At the end of the experiment, the LV mass index (LVMI) was evaluated. In addition, LV structure and function, collagen content, microstructure and ultrastructure were examined using echocardiography, the hydroxyproline assay, routine light microscopy and transmission electron microscopy, respectively. In the losartan- and GCIP-27 (10, 30 and 90 microg/kg)-treated groups, SBP was decreased significantly compared with that of the vehicle (saline) group. However, even at the highest concentration used, the hypotensive effect of GCIP-27 was weaker than that of losartan. For example, after 8 weeks treatment, SBP had decreased by 30.4% in the losartan-treated group compared with decreases of 10.5, 13.1 and 18.5% in the 10, 30 and 90 microg/kg GCIP-27-treated groups, respectively. Both GCIP-27 (10, 30 and 90 microg/kg) and losartan (6 mg/kg) significantly reduced LV posterior wall thickness, the thickness of the interventricular septum, collagen content and LVMI, with the effects of GCIP-27 at all three concentrations tested being greater than those of losartan. In conclusion, GCIP-27 effectively attenuates LV remodelling in SHR and the antiremodelling effect may not be dependent entirely on decreases in blood pressure.